Our goal is to investigate expression of Toll-like receptors (TLR) on human conjunctival epithelium and determine the potential contributions of TLR activation by microbial ligands to ocular allergic inflammation. Studies examining conjunctival epithelial cells as immunomodulators have been focused on the effects of pro-inflammatory cytokines, while the direct effects of microbial products have not been examined. Preliminary studies suggest that conjunctival epithelial cells express a pattern recognition receptor, TLR2, and respond to Staphylococcus aureus peptidoglycan (SA-PGN) with mediator release and surface receptor upregulation. It is likely that simultaneous activation of conjunctival epithelial cells via TLRs and mast cell mediators is a frequent occurrence, which may contribute to the development of chronic ocular allergic disease. The first specific aim will focus on determining the repertoire of TLRs (focusing on TLR1,2,4,6,9 and accessory receptor, CD14) expressed by conjunctival epithelial cells and whether the TLRs expressed are inducible by cytokines and/or TLR ligands (SA-PGN and yeast zymosan for TLRs 1,2, and 6; Pseudomonas aeruginosa LPS for TLR4, CpG DNA for TLR9). This will include in vivo examination of cells obtained from the ocular surface of human subjects (normal vs various disease states) via impression cytology. The second specific aim will investigate activation of conjunctival epithelial cells with TLR ligands, specific binding of microbial products to their respective TLRs and signaling (accessory proteins MyD88, MD2, and TIRAP). Activation will be defined as upregulation of ICAM-1 and/or HLA-DR expression and/or enhanced release of TNFalpha, IL-6, IL-8, and/or RANTES. The third specific aim will examine the effect of simultaneous and sequential activation of conjunctival epithelial cells with TLR ligands and supernates from IgE-activated purified conjunctival mast cells. The results of combined activation will be compared to activation with either TLR ligands or IgE-activated mast cell supernates alone. Conjunctival epithelial and mast cells will be obtained from cadaveric conjunctival tissues. Techniques to evaluate protein expression will include flow cytometry, Western blot, Immuno-PCR (in impression cytology cells), and ELISA. Techniques to evaluate mRNA expression will include Northern blot and RT-PCR (in impression cytology cells). Techniques to examine binding specificity and signaling complexes will include blocking antibodies, co-immunoprecipitation and gel shift assays.